Obesity is a risk factor for asthma, but standard asthma drugs have reduced efficacy in obese asthmatics. Understanding the mechanistic basis for obesity-related asthma could lead to new treatments for these patients. Obese mice exhibit innate airway hyperresponsiveness (AHR), a characteristic of asthma, and also develop greater increases in airway responsiveness after acute exposure to ozone (O3), an asthma trigger. Alterations in the gut microbiome occur in obesity, contribute to some obesity-related conditions, including insulin resistance, and may also lead to the innate AHR and the augmented O3-induced AHR of obesity. The metabolome is the set of small molecule metabolites present in a given biological fluid or tissue. The metabolomes of the liver, serum, urine, and adipose tissue are altered in obesity. Our data indicate that the lung metabolome is also affected by obesity and by O3. The microbiome impacts the metabolomes of intestines, urine, liver, brain, and kidney. Our preliminary data also indicate alterations in bacterial-derived metabolites in the lung with obesity and with O3 exposure. Thus, the altered microbiome of obesity may change metabolites present in the lung that can affect airway function. Indeed preliminary data indicate reduced O3-induced AHR in mice treated with antibiotics. Hence, our hypothesis is obesity-related changes in the microbiome and consequent effects on the lung metabolome contribute to the asthma- like phenotype of obese mice. We propose 2 specific aims. In aim 1, we will treat obese and lean mice with a cocktail of antibiotics or with regular drinking water for two weeks. Other lean mice treated with antibiotics will have their microbiota reconstituted by oral gavage of feces derived from obese or lean mice. Mice will then be exposed to room air or to O3 followed by measurements of airway responsiveness, bronchoalveolar lavage (BAL), and tissue harvest. Fecal pellets will be collected to confirm 1) obesity-related changes in the microbiome and 2) the efficacy of the antibiotic treatment and fecal transplant. If obesity-related changes in the microbiome contribute to the asthma-like phenotype of obese mice, we will observe that the innate AHR and the O3-induced AHR are attenuated after antibiotic treatment and restored by oral gavage of obese feces. In aim 2, we will perform a metabolomic analysis of lung tissue harvested from mice treated as in aim 1. We expect that after antibiotic treatment, obesity-related changes in the lung metabolome and in the impact of O3 on the lung metabolome will be reduced. 16S pyro sequencing of BAL fluid will also be used to determine if there are effects of obesity, O3, or oral antibiotics on the lung microbiome. Impact: These studies are expected to provide proof of concept that the asthma-like phenotype of obese mice is affected by their microbiome, setting the stage for future studies of the therapeutic efficacy of probiotics and prebiotics. These studies will also identify lung metabolites that track with obesity-related AHR. Future studies would examine whether such metabolites can be targeted therapeutically or used diagnostically.